. This is a competitive renewal of an ROI research project grant. The aim of the proposed research is to apply state-of-the-art rheological methods, fluorescent optical sectioning microscopy and cell and molecular biology techniques to the study of atherosclerosis and thrombosis. Four specific aims are outlined: 1) To understand the observed differences in gene expression patterns in endothelial cells (Ecs) and smooth muscle cells (SMCs) produced by physiological levels of fluid shear stress and cyclical mechanical strain. 2) To interpret the effect of shear preconditioning on gene expression profiles in response to subsequent change in mechanical environment or cytokine stimulation. 3) To understand the potential differences between various endothelial cell types in their response to mechanical forces. 4) To interpret early signal transduction of flow induced shear stress to the cellular nucleus via either cytoskeletal connections and/or diffusible second messengers. The PI hypothesizes that both stress and strain have quite different effects on gene expression and regulation of cell phenotype. Proteins important in thrombosis and atherosclerosis (thrombin receptor 1 [protease activated receptor - 1 or PAR - 1], VEGF, PGH Synthases, ICAM, VCAM, GP1b [a platelet receptor that can be expressed on endothelial cells] and von Willebrand factor) have been targeted for study. The PI has shown previously dramatic effects of arterial shear stress on TPA and endothelin syntheses and secretion. In addition, new DNA micro array technology will be used to greatly increase the amount of information obtained per experiment. This approach could lead to an understanding of groups of genes that are regulated in a coordinated manner by mechanical forces and thus result in important new hypotheses in vascular biology. A major question is how are mechanical signals transduced to produce intracellular changes. Optical sectioning fluorescence microscopy and digital image processing will be used to study stress effects on calcium fluxes and intracellular pH. The equipment is able to obtain both multicellular averages and single cell changes in fluorescence. The ability has also been developed to examine the three-dimensional distribution of ions in a single-cell using optical sectioning and image analysis and reconstruction. Changes in nuclear geometry in response to cell mechanical loading will also be examined using this technology. A final question to be examined is the relative role of stress (or strain) magnitude versus the rate of change of stress (or strain). Since arterial blood flow is inherently pulsatile, in vivo application of the PI's in vitro results will require this knowledge.